AIM: To investigate the correlation between expression of calreticulin and infiltration of lymphocytes in stage ⅢB colon cancer. METHODS: Sixty-eight pathologically-confirmed speci-mens were obtained from stage ⅢB (...AIM: To investigate the correlation between expression of calreticulin and infiltration of lymphocytes in stage ⅢB colon cancer. METHODS: Sixty-eight pathologically-confirmed speci-mens were obtained from stage ⅢB (T3N1M0) colon cancer patients who underwent radical resection between January 1999 and May 2002 at the Cancer Center of Sun Yat-Sen University, Guangzhou, China. Immuno-histochemical analysis was performed to show infiltration of lymphocytes and expression of calreticulin in colon cancer. Association between calreticulin expression, infiltration of lymphocytes, and 5-year survival rate of patients was assessed. RESULTS: The expression level of calreticulin was lower in cancer nest than in its adjacent normal epithelium since 61.8% (42/68) of the samples were stained with calreticulin in colon cancer. The expression of calreticulin in colon cancer was associated with the infi ltration of CD45RO+ cells rather than with that of CD3+ cells. In addition, the stronger expression of calreticulin and the higher infiltration of CD3+ and CD45RO+ cells in colon cancer were associated with the higher 5-year survival rate of patients. CONCLUSION: Expression of calreticulin is associated with infiltration of T-cells, which implies that a low expression level of molecular marker may represent a new mechanism underlying immune escape in colon cancer.展开更多
AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HB...AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.展开更多
Objective: To investigate the recombinant calreticulin (rCRT) mediated antitumor immune response. Methods: Cell proliferation was determined by MTT method, apoptosis was evaluated by DNA fragmentation and CRT expressi...Objective: To investigate the recombinant calreticulin (rCRT) mediated antitumor immune response. Methods: Cell proliferation was determined by MTT method, apoptosis was evaluated by DNA fragmentation and CRT expression and cell localization were assayed by western blotting, QT-RT-PCR and immunofluorescence assays. The mouse melanoma cell line B16-F1 was treated with polyamine analogue BENS to induce apoptosis and incubated with rCRT to get rCRT coated on the membrane, and then the cells were used to immune BALB/c mice as a cell-antigen. Immunized animals were rechallenged by live B16-F1 cells and then tumor generation ratio and the lactate dehydrogenase release assay were used to evaluate antitumor effects of rCRT-mediated immunity. Results: BENS induced apoptosis of B16-F1 cells without the redistribution of CRT within the cells. When B16-F1 cells coated with rCRT were used as cell-antigen to inoculate the animals, the mice obtained the ability in inhibiting proliferation of homologous tumor cells in vivo. Comparing with the positive control group, the splenocytes from those inoculated mice have an obvious enhancement on their cytolytic effects specifically against B16-F1 cells. Conclusion: rCRT coated on the cell surface can enhance immunogenicity of apoptotic tumor cells and mediated effective anti-tumor immunoresponse in mice.展开更多
Calreticulin(CRT)is a highly conserved Ca2+-binding protein and chaperone in the endoplasmic reticulum,which mainly participates in adjusting calcium level and directing proper conformation of proteins.Here,we cloned ...Calreticulin(CRT)is a highly conserved Ca2+-binding protein and chaperone in the endoplasmic reticulum,which mainly participates in adjusting calcium level and directing proper conformation of proteins.Here,we cloned the crt gene of the scleractinian coral Galaxea astreata,named Gacrt,and analysed its ability to drive bacterial agglutination.The full-length Gacrt cDNA consisted of 1792 nucleotides and contained a 77 bp 5’untranslated region(UTR),a 380 bp 3’UTR and a 1335 bp open reading frame(ORF)that encoded a 444 amino acid protein.The deduced peptide possessed a signal peptide domain,an endoplasmic reticulum retrieval signal sequence(KDEL),two potential calreticulin family signature motifs and a set of triplicate repeats.We also found that the recombinant GaCRT protein could promote agglutination of both the Gram-positive bacterium Micrococcus luteus and the Gram-negative bacterium Escherichia coli.These results show that the GaCRT protein can enhance bacterial agglutination,hinting that GaCRT is an immune-relevant molecule involved in host defense against bacterium.展开更多
Calreticulin(CRT)is a multifunctional molecule in both intracellular and extracellular environment.We have previ-ously found that a recombinant CRT fragment(rCRT/39-272)could modulate T cell-mediated immunity in mice ...Calreticulin(CRT)is a multifunctional molecule in both intracellular and extracellular environment.We have previ-ously found that a recombinant CRT fragment(rCRT/39-272)could modulate T cell-mediated immunity in mice via activation and expansion of CD1dhiCD5+B cells as well as induction of CRT-specifi c regulatory antibodies.Anti-body secreting cells(ASCs)are terminally differentiated B cells responsible for producing antibodies to participate in positive immune response as well as immune regula-tion.In this study,we demonstrate that rCRT/39-272 dif-ferentiates murine CD1dhiCD5+B cells into ASCs mark-ed by increased expression of plasma cell-associated transcription factors and production of polyreactive antibodies against DNA and CRT in vitro.Intraperitoneal administration of rCRT/39-272 augmented differentiation of CD1dhiCD5+B cells into ASCs in naïve mice or mice with experimental autoimmune encephalomyelitis.Thus,we propose that ASC differentiation and subsequent an-tibody production of CD1dhiCD5+B cells are key steps in CRT-mediated immunoregulation on infl ammatory T cell responses.展开更多
Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralizati...Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium. All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.展开更多
BACKGROUND There is an intimate crosstalk between cancer formation,dissemination,treatment response and the host immune system,with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatme...BACKGROUND There is an intimate crosstalk between cancer formation,dissemination,treatment response and the host immune system,with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatments.However,inducing a purposeful synergistic response between conventional therapies and the immune system remains evasive.The release of damage associated molecular patterns(DAMPs)is indicative of immunogenic cell death and propagation of established immune responses.However,there is a gap in the literature regarding the importance of DAMP expression in oesophageal adenocarcinoma(OAC)or by immune cells themselves.AIM To investigate the effects of conventional therapies on DAMP expression and to determine whether OAC is an immunogenic cancer.METHODS We investigated the levels of immunogenic cell death-associated DAMPs,calreticulin(CRT)and HMGB1 using an OAC isogenic model of radioresistance.DAMP expression was also assessed directly using ex vivo cancer patient T cells(n=10)and within tumour biopsies(n=9)both pre and post-treatment with clinically relevant chemo(radio)therapeutics.RESULTS Hypoxia in combination with nutrient deprivation significantly reduces DAMP expression by OAC cells in vitro.Significantly increased frequencies of T cell DAMP expression in OAC patients were observed following chemo-(radio)therapy,which was significantly higher in tumour tissue compared with peripheral blood.Patients with high expression of HMGB1 had a significantly better tumour regression grade(TRG 1-2)compared to low expressors.CONCLUSION In conclusion,OAC expresses an immunogenic phenotype with two distinct subgroups of high and low DAMP expressors,which correlated with tumour regression grade and lymphatic invasion.It also identifies DAMPs namely CRT and HMGB1 as potential promising biomarkers in predicting good pathological responses to conventional chemo(radio)therapies currently used in the multimodal management of locally advanced disease.展开更多
基金Supported by National Nature Science Foundation of China, 30972882Key Projects of Science and Technology of Guang-dong Province, China, 2008B030301079
文摘AIM: To investigate the correlation between expression of calreticulin and infiltration of lymphocytes in stage ⅢB colon cancer. METHODS: Sixty-eight pathologically-confirmed speci-mens were obtained from stage ⅢB (T3N1M0) colon cancer patients who underwent radical resection between January 1999 and May 2002 at the Cancer Center of Sun Yat-Sen University, Guangzhou, China. Immuno-histochemical analysis was performed to show infiltration of lymphocytes and expression of calreticulin in colon cancer. Association between calreticulin expression, infiltration of lymphocytes, and 5-year survival rate of patients was assessed. RESULTS: The expression level of calreticulin was lower in cancer nest than in its adjacent normal epithelium since 61.8% (42/68) of the samples were stained with calreticulin in colon cancer. The expression of calreticulin in colon cancer was associated with the infi ltration of CD45RO+ cells rather than with that of CD3+ cells. In addition, the stronger expression of calreticulin and the higher infiltration of CD3+ and CD45RO+ cells in colon cancer were associated with the higher 5-year survival rate of patients. CONCLUSION: Expression of calreticulin is associated with infiltration of T-cells, which implies that a low expression level of molecular marker may represent a new mechanism underlying immune escape in colon cancer.
基金Supported by Grants from National Natural Science Foundation of China, No. 30901344
文摘AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.
基金supported by a grant from the National Natural Science Foundation of China (No.30973445)
文摘Objective: To investigate the recombinant calreticulin (rCRT) mediated antitumor immune response. Methods: Cell proliferation was determined by MTT method, apoptosis was evaluated by DNA fragmentation and CRT expression and cell localization were assayed by western blotting, QT-RT-PCR and immunofluorescence assays. The mouse melanoma cell line B16-F1 was treated with polyamine analogue BENS to induce apoptosis and incubated with rCRT to get rCRT coated on the membrane, and then the cells were used to immune BALB/c mice as a cell-antigen. Immunized animals were rechallenged by live B16-F1 cells and then tumor generation ratio and the lactate dehydrogenase release assay were used to evaluate antitumor effects of rCRT-mediated immunity. Results: BENS induced apoptosis of B16-F1 cells without the redistribution of CRT within the cells. When B16-F1 cells coated with rCRT were used as cell-antigen to inoculate the animals, the mice obtained the ability in inhibiting proliferation of homologous tumor cells in vivo. Comparing with the positive control group, the splenocytes from those inoculated mice have an obvious enhancement on their cytolytic effects specifically against B16-F1 cells. Conclusion: rCRT coated on the cell surface can enhance immunogenicity of apoptotic tumor cells and mediated effective anti-tumor immunoresponse in mice.
基金The National Marine Welfare Industry Research Project under contract No.201105012the Guangdong Provincial Natural Science Foundation under contract No.S2011010000269+1 种基金the Guangdong Marine Fishery Science and Technology Extension Project under contract No.A201308E02the Guangdong Ocean University Scientific Research Start-up Fund for the Doctoral Program under contract No.R17066。
文摘Calreticulin(CRT)is a highly conserved Ca2+-binding protein and chaperone in the endoplasmic reticulum,which mainly participates in adjusting calcium level and directing proper conformation of proteins.Here,we cloned the crt gene of the scleractinian coral Galaxea astreata,named Gacrt,and analysed its ability to drive bacterial agglutination.The full-length Gacrt cDNA consisted of 1792 nucleotides and contained a 77 bp 5’untranslated region(UTR),a 380 bp 3’UTR and a 1335 bp open reading frame(ORF)that encoded a 444 amino acid protein.The deduced peptide possessed a signal peptide domain,an endoplasmic reticulum retrieval signal sequence(KDEL),two potential calreticulin family signature motifs and a set of triplicate repeats.We also found that the recombinant GaCRT protein could promote agglutination of both the Gram-positive bacterium Micrococcus luteus and the Gram-negative bacterium Escherichia coli.These results show that the GaCRT protein can enhance bacterial agglutination,hinting that GaCRT is an immune-relevant molecule involved in host defense against bacterium.
基金This study was supported by grants from PCSIRT(IRT1075)the National Natural Science Foundation of China(Grant Nos.31370908,31070781,and 31100633)+1 种基金the National Basic Research Program(973 Program)(No.2010CB529102)Foundation of Nature Science of Jiangsu Higher Education Institutions of China(11KJB180011).
文摘Calreticulin(CRT)is a multifunctional molecule in both intracellular and extracellular environment.We have previ-ously found that a recombinant CRT fragment(rCRT/39-272)could modulate T cell-mediated immunity in mice via activation and expansion of CD1dhiCD5+B cells as well as induction of CRT-specifi c regulatory antibodies.Anti-body secreting cells(ASCs)are terminally differentiated B cells responsible for producing antibodies to participate in positive immune response as well as immune regula-tion.In this study,we demonstrate that rCRT/39-272 dif-ferentiates murine CD1dhiCD5+B cells into ASCs mark-ed by increased expression of plasma cell-associated transcription factors and production of polyreactive antibodies against DNA and CRT in vitro.Intraperitoneal administration of rCRT/39-272 augmented differentiation of CD1dhiCD5+B cells into ASCs in naïve mice or mice with experimental autoimmune encephalomyelitis.Thus,we propose that ASC differentiation and subsequent an-tibody production of CD1dhiCD5+B cells are key steps in CRT-mediated immunoregulation on infl ammatory T cell responses.
基金the National Natural Science Foundation of China (Nos. 30371092, 30221003, and 30530600)
文摘Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium. All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.
文摘BACKGROUND There is an intimate crosstalk between cancer formation,dissemination,treatment response and the host immune system,with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatments.However,inducing a purposeful synergistic response between conventional therapies and the immune system remains evasive.The release of damage associated molecular patterns(DAMPs)is indicative of immunogenic cell death and propagation of established immune responses.However,there is a gap in the literature regarding the importance of DAMP expression in oesophageal adenocarcinoma(OAC)or by immune cells themselves.AIM To investigate the effects of conventional therapies on DAMP expression and to determine whether OAC is an immunogenic cancer.METHODS We investigated the levels of immunogenic cell death-associated DAMPs,calreticulin(CRT)and HMGB1 using an OAC isogenic model of radioresistance.DAMP expression was also assessed directly using ex vivo cancer patient T cells(n=10)and within tumour biopsies(n=9)both pre and post-treatment with clinically relevant chemo(radio)therapeutics.RESULTS Hypoxia in combination with nutrient deprivation significantly reduces DAMP expression by OAC cells in vitro.Significantly increased frequencies of T cell DAMP expression in OAC patients were observed following chemo-(radio)therapy,which was significantly higher in tumour tissue compared with peripheral blood.Patients with high expression of HMGB1 had a significantly better tumour regression grade(TRG 1-2)compared to low expressors.CONCLUSION In conclusion,OAC expresses an immunogenic phenotype with two distinct subgroups of high and low DAMP expressors,which correlated with tumour regression grade and lymphatic invasion.It also identifies DAMPs namely CRT and HMGB1 as potential promising biomarkers in predicting good pathological responses to conventional chemo(radio)therapies currently used in the multimodal management of locally advanced disease.
